Skeletal muscle trauma to the head or neck can not only be debilitating, but can result in severe psychological impairment. While skeletal muscle has an innate repair mechanism directed by growth factors and mediated by satellite cells, this mechanism cannot compensate for large, volumetric muscle loss as a result of trauma related events such as car accidents or cancer resection. Clinical strategies to treat these defects consist of autologous tissue transfer techniques, requiring large donor sites and extensive surgical procedures that can result in donor site morbidity and limited functional recovery. As such, there is a clinical need for an off-the-shelf, bioactive scaffold that directs patient's cell to align and differentiate into muscle tissue in situ. Our laboratory recently developed fibrin microthreads, a tissue construct that resembles the morphologic structure of a muscle fiber and can align cells longitudinally along the fiber length. In the wound site, fibrin degrades quickly i the presence of numerous proteinases such as plasmin. We recently developed a crosslinking strategy to modify fibrin microthreads to increase their resistance to enzymatic degradation. The overall goal of this project is to use these microthreads to design a novel biomimetic scaffold that will utilize the endogenous skeletal muscle regeneration pathway to regenerate volumetric muscle loss. We hypothesize that fibrin microthreads, with precisely tuned degradation properties and conjugated with wound healing growth factors such as hepatocyte growth factor (HGF) and insulin-like growth factor (IGF)-I, will enhance skeletal muscle regeneration of a large muscle defect via endogenous pathways. To systematically test this hypothesis, we will analyze the effects of synthetic and natural crosslinking agents on the structural and biochemical properties of fibrin microthreads. We will analyze the dose-dependent responses of satellite cells to growth factors on fibrin microthreads that contribute to the skeletal muscle regeneration pathway and determine how these factors affect cell phenotype through immunohistochemical assays. We will assess the contribution of fibrin microthreads to regeneration using a murine muscle excision model to analyze the recovery of mechanical function and collagen deposition within the wound site over time. Our ultimate goal is to develop a scaffold that will persist in siu throughout the peak time of skeletal muscle regeneration, 2-4 weeks, with clinically relevant growth factor concentrations to direct mechanically functional tissue regeneration in vivo through the study of the following Specific Aims: (1) To develop microthreads with structural and biochemical properties similar to native skeletal muscle, and (2) assess microthreads' enhancement of skeletal muscle regeneration in vivo. Through the combination of finely tuned structural and mechanical properties, and the conjugation of growth factors designed to promote and mimic skeletal muscle regeneration, we will create a biomimetic scaffold to regenerate large skeletal muscle defects in vivo.